Selected reaction monitoring approach for validating peptide biomarkers

Abstract

We here describe a selected reaction monitoring (SRM)-based approach for the discovery and validation of peptide biomarkers for cancer. The first stage of this approach is the direct identification of candidate peptides through comparison of proteolytic peptides derived from the plasma of cancer patients or healthy individuals. Several hundred candidate peptides were identified through this method, providing challenges for choosing and validating the small number of peptides that might prove diagnostically useful. To accomplish this validation, we used 2D chromatography coupled with SRM of candidate peptides. We applied this approach, called sequential analysis of fractionated eluates by SRM (SAFE-SRM), to plasma from cancer patients and discovered two peptides encoded by the peptidyl-prolyl isomerase A (PPIA) gene whose abundance was increased in the plasma of ovarian cancer patients. At optimal thresholds, elevated levels of at least one of these two peptides was detected in 43 (68.3%) of 63 women with ovarian cancer but in none of 50 healthy controls. In addition to providing a potential biomarker for ovarian cancer, this approach is generally applicable to the discovery of peptides characteristic of various disease states.

A Multiplex Assay for the Stratification of Patients with Primary Central Nervous System Lymphoma Using Targeted Mass Spectrometry

“…From these candidate biomarkers, we selected 33 candidate peptide biomarkers for further evaluation. As targeted mass spectrometric analysis offers high specificity for peptide identification and quantification in complex protein mixtures as well as direct transferability for candidate validation, we decided to consider selected reaction monitoring (SRM) analysis [19].…”

Direct Detection and Quantification of Neoantigens

“…An HPLC fractionation was performed to separate the neoantigenic peptides into 96 fractions based on each peptide's hydrophobicity in a weak basic environment (pH ¼ 8.2). The bRPLC solvents were used for this HPLC separation performed as previously described (19). Neoantigenic peptides were then organized into 32 groups comprising three sequential fractions each, according to the MANA-SRM fraction group scheme as previously described (19).…”

“…The bRPLC solvents were used for this HPLC separation performed as previously described (19). Neoantigenic peptides were then organized into 32 groups comprising three sequential fractions each, according to the MANA-SRM fraction group scheme as previously described (19). All MANA-SRM fractions of neoantigenic peptides were dried in a Speed-Vac at 35 C with vacuum pressure below 5 mbar.…”

“…The dissolved peptide sample was reduced again by tris(2-carboxyethyl)phosphine (TCEP) at 10 mmol/L at low pH (pH ¼ 3) followed by an incubation at 60 C for 15 minutes. The reduced samples were ready for MANA-SRM mass spectrometry assays as previously described (19).…”

Targeted Omics: Finding the Needle

“…Instrumental developments led to the scanning speed of triple quadrupole instruments to increase remarkably over the last 10 years, facilitating today the measurement of more than 600 transitions per second and over 200 protein targets on a modern triple quadrupole instrument. [ 11,12 ] Using the ion‐funnel technology to focus the entering ion beam, further improved the sensitivity of targeted assays. [ 13 ] However, triple quadrupole‐based targeted proteomics is still a low‐resolution approach to analyze peptides.…”